Many plasmid vectors are currently available for expression in animal cells. Commercial providers of such vectors include Invitrogen (Carlsbad, Calif.), Promega (Madison, Wis.) and Clontech (Mountain View, Calif.). In general, the two key elements in these vectors are a strong promoter and a convenient multiple cloning site (MCS) for insertion of genes of interest. Using such vectors, transient expression of the target gene can be readily achieved in short term cultures.
However, in order to achieve sustained high levels of gene expression, vectors are preferred that contain a selectable marker to grow and maintain stable transfectants. In order to achieve replication and propagation in daughter cells, the recombinant plasmid must integrate into the host genome after transfection. This event is relatively rare and rate limiting as well as subject to the vagaries of each particular site of integration (Al Shawi R., Kinnaird J., Burke J. and Bishop J. O. 1990. Expression of a foreign gene in a line of transgenic mice is modulated by a chromosomal position effect. Mol. Cell. Biol. 10:1192-1198).
To circumvent the consequences of genomic integration altogether, vectors capable of autonomous replication and adequate segregation during cell division can be employed. This vector category includes Mammalian Artificial Chromosomes (MACs) (Lindenbaum M, Perkins E, Csonka E, Fleming E, Garcia L, Greene A, Gung L, Hadlaczky G, Lee E, Leung J, MacDonald N, Maxwell A, Mills K, Monteith D, Perez C F, Shellard J, Stewart S, Stodola T, Vandenborre D, Vanderbyl S, Ledebur HC Jr. 2004. A mammalian artificial chromosome engineering system (ACE System) applicable to biopharmaceutical protein production, transgenesis and gene-based cell therapy. Nucleic Acids Res. 32(21):e172) and episomal vectors such as plasmids derived from the Epstein-Barr virus (EBV). EBV-derived plasmids can be stably maintained in dividing cells through the use of the viral replication and segregation elements (Yates, J. L., Warren, N., and Sugden, B. 1985. Stable replication of plasmids derived from Epstein-Barr virus in various mammalian cells. Nature 313:812-815).
An advantage of episomal vectors over MACs is that they are maintained at multiple copies per cell (Conese, M., Auriche, C., and Ascenzioni, F. 2004. Gene therapy progress and prospects: episomally maintained self-replicating systems. Gene Ther. 24:1735-1741), thus naturally enhancing expression levels. The relatively smaller EBV-derived plasmids (10-100 kb in size) can also be shuttled from the mammalian host cell back into bacteria for analysis or propagation (Kelleher Z. T., Fu H., Livanos E., Wendelburg B., Gulino S. and Vos J. M. 1998. Epstein-Barr-based episomal chromosomes shuttle 100 kb of self-replicating circular human DNA in mouse cells. Nat. Biotechnol. 16:762-768; Wade-Martins R., Frampton J. and James M. R. 1999. Long-term stability of large insert genomic DNA episomal shuttle vectors in human cells. Nucleic Acids Res. 27:1674-1682). In contrast, the relatively larger MACs (>1 Mb in size) have to be propagated in suitable mammalian host cells. Finally, plasmids can be more readily defined in that their entire sequence can be determined at each step of development using standard techniques and molecular biology tools. In contrast, only a small percentage of a MAC can be known with certainty at any point by direct sequencing, and structural analysis of MACs requires sophisticated techniques such as flourescence in situ hybridization (FISH) and flow cytometry.
There is currently a need for more flexible but powerful expression vectors to address an expanding market for complex glycosylated proteins. There is also a need for efficient non-viral gene delivery systems for many transgenic applications and in gene therapy. The disclosed invention, a novel episomal vector design, has the desired characteristics to meet these needs.